We SB-505124 HCl repeated the same treatment (RAD001 plus MK-2206 combination) in SGC-7901, GES-1 cells (high PTEN expression) and MKN-28 cells(middle PTEN expression). Results in Physique S1E showed that RAD001 and MK-2206 synergistically induced HGC-27 cell death, as the number of trypan blue cells (lifeless cells) increased significantly after the co-administration, comparable results were also obtained in SNU-601 cells (data not shown). Hence, the combination of RAD001 and MK-2206 showed synergistic inhibitory activity on HGC-27 and SNU-601 cell growth in vitro.
#Calcusyn online software
The computer software Calcusyn was used to test combination index (CI) between RAD001 and MK-2206, CI<1.0 was considered as synergism, as seen in Physique S1C and D. Further, RAD001 and MK-2206 at ratio 110 showed most significant synergistic effects SB-505124 HCl (Physique 2A and B). CCK-8 cell viability results in Physique 2A and B exhibited that either RAD001 or MK-2206 alone experienced a moderate effect on HGC-27 and SNU-601 cell growth, however, combination of the two at a relative lower concentration significantly inhibited the growth of both cells, as the CCK-8 OD value decreased dramatically in cells treated with both brokers (Physique 2A and B). RAD001 and MK-2206 synergistically inhibits the growth of HGC-27 and SNU-601 cells The main object of this current study is usually to test the synergistic anti-gastric malignancy cell ability of RAD001 and MK-2206. The data shown was the mean from three impartial experiments. AGS and HGC-27 cells were treated with different concentration of RAD001 for 24 hours, phospho- and total S6 were detected by Western blot, pS6 level was quantified as explained (D and E), and the number was normalized to the band labeled with 1.00. The expression level of PTEN, pAKT (Ser 473) and -actin (equivalent loading) in above cell lines and GES-1 cells were also detected by western blot, PTEN and pAKT level was quantified as explained (C). IC-50 of RAD001 in these cell lines was shown (B). Open in a separate window SB-505124 HCl Physique 1 RAD001 inhibits cell growth in multiple human gastric malignancy cell lines.Cultured gastric cancer cell lines AGS, MNK-45, HGC-27, SNU-601, MKN-28, SGC-7901 and N-87 were treated with different concentration of RAD001 for 72 h, SB-505124 HCl afterwards, cell growth was detected by CCK-8 cell viability assay (A). More ever, HGC-27 and AGS were both sensitive to RAD001 on mTOR (pS6) inhibition (Physique 1D and E). Results indicated that RAD001-sensitive lines were cells with no or low expression level of PTEN (HGC and SNU601).
Results in Physique 1C show that SNU-601 cells expressed extremely low level of PTEN, which was also supported by paper by Byun DS et al.
Western blot results in Physique 1C showed the expression of PTEN and p-AKT (Ser 473) in above gastric malignancy cells. HGC-27 and SNU-601 experienced the lowest IC-50, which further confirmed their highest sensitivity to RAD001 (Physique 1B,). IC-50s of RAD001 in these different lines were also offered. HGC-27 and SNU-601,were the most sensitive ones (Physique 1A, Physique S1A and B).
However, these different lines showed different sensitivity to RAD001. RAD001 inhibited cell growth in all gastric malignancy cells, as the cell viability OD decreased after different concentrations of RAD001 treatment (Physique 1A, Figure S1A and B). Gastric malignancy cell growth was reflected by cell viability which was detected by CCK-8 assay. Results RAD001 inhibits cell growth in multiple human gastric malignancy cell lines We first examined the activity of RAD001 on cell growth in gastric malignancy cell lines representing different genetic backgrounds, including AGS, MNK-45, HGC-27, SNU-601, MKN-28, SGC-7901 and N-87. CalcuSyn software (Version 2.0) was obtained from (Beijing, China), and combination index (CI)<1 indicates synergism. *followed by multiple comparisons performed with post hoc Bonferroni test (SPSS version 14). The Calcusyn software was put on calculate combination index (CI) between each RAD001 and/or MK-2206 dose combination for the CCK-8 data obtained from HGC-27,SNU-601 and GES-1 cells (see Figure 2A, B and E).
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